Embryonic stem cells, such as mesenchymal stem cells, are generally considered the most desirable type of pluripotent cells useful for research and therapeutic use. However, there are religious and ethical objections to the use of embryonic stem cells, and federal funding of research utilizing embryonic stem cells has been restricted to cells from 14 embryonic cell lines. Some researchers fear one or more of the federally approved embryonic stem cell lines may become contaminated or non-viable.
In response, researchers have sought non-embryonic stem cell sources. Umbilical cord and placenta are believed to be rich sources of stem cells. See Dhot et al., “Cord blood stem cell banking and transplantation,” Indian J. Pediatr. 70:989-992 (2003) and “Umbilical Cord Matrix, a rich new stem cell source, study shows,” Life Science News (Jan. 16, 2005).
An advantage of harvesting umbilical cord and placenta for stem cells is that a far greater number of stem cells can be recovered from the umbilical cord and placenta than from an embryo. More particularly, only about 30 to 35 stem cells can be obtained per embryo. In comparison, about 10.1+/−1.2 108 stem cells can be extracted from an umbilical cord, and about 7.1+/−0.8 108 stem cells can be extracted from a placenta, including the umbilical cord. Barney, “The daily interview: the market opportunity for stem cell research,” TheStreet.com (Aug. 6, 2001).
Umbilical cord and placental stem cells can also be cultured to grow more stem cells after cryopreservation. Liu et al., “Cryobiological characteristics of placental cord blood preserved in bioarchive auto-preserved liquid nitrogen system,” Zhongguo Shi Yan Xue Ye Xue Za Zhi., 10:261-264 (2002).
There are at least two methods typically used to obtain stem cells from the umbilical cord or placenta. The first method involves simply draining blood from the placenta and/or umbilical cord into a closed sterile collection bag using gravity. Solves et al., “Comparison between two strategies for umbilical cord blood collection,” Bone Marrow Transplant. 31:269-273 (2003). Other researchers have used pressure to extract blood from the umbilical cord and/or placenta. See, for example, Romanov et al., “Searching for alternative sources of postnatal human mesenchymal stem cells: candidate MSC-like cells from umbilical cord,” Stem Cells 21:105-110 (2003) (Umbilical cord vein cannulated on both sides, washed with Earle's balanced salt solution, and then gently “massaged” to collect a suspension of endothelial and subendothelial cells).
An important potential use for umbilical and placental stem cells is for unrelated bone marrow donor/recipient transplantation. Stevens et al., “Placental/umbilical cord blood for unrelated-donor bone marrow reconstitution: relevance of nucleated red blood cells,” Blood, 100:2662-2664 (2002). However, the yield from the umbilical cord is only sufficient for pediatric transplantation. When researchers attempted to transplant umbilical cord stem cells into adults, the procedure was unsuccessful due to insufficient stem cell yields.
Perfusion science seeks to maintain an organ's natural function using mechanical means. Perfusion has been mostly utilized in cardiac-thoracic surgery, vascular surgery, and preservation of organs for transplantation. See, for example, U.S. Pat. No. 6,811,965.
At least one researcher has flushed the placenta with perfusate through the arterial-vein circuit to eliminate tissue residual blood, Zhang et al., “Human placenta-derived mesenchymal progenitor cells support culture expansion of long-term culture-initiating cells from cord blood CD34+ cells,” Exp Hematol. 32:657-664 (2004). However, there have been no reported extractions of cord blood from either an umbilical cord or a placenta using pulsatile perfusion.
There are significant differences between pulsatile perfusion of the placenta and non-pulsatile perfusion, which can include the following:
1. pulsatile perfusion mimics the action of the heart, thus allowing for a smooth transition from the mother to the perfusion circuit;
2. pulsatile perfusion has been shown to vasodilate the vascular structure of organs and also vasodilates the placental vascular structure;
3. pulsatile perfusion increases the osmotic pressure of the perfusion solution, thus more efficiently removing the placental blood from the interior of the placental cells;
4. in pulsatile perfusion one typically adjusts the perfusate chemistries, pH, PCO2 and PO2, to duplicate normal body chemistries, thus extracting placental blood without causing any detrimental effects, such as renewed vasoconstriction of the vascular structure;
5. pulsatile perfusion is less harmful to the endothelial cells of the vascular structure, thus allowing placental arteries and vein to be used for human vascular allografts.
An object of this invention is to provide an improved method for obtaining pluripotent stem cells without destroying an embryo. Another object of this invention is to provide a method for obtaining pluripotent stem cells in sufficient yield to permit unrelated adult bone marrow transplants of such pluripotent stem cells.
A feature of this invention is the use of pulsatile perfusion to extract stem cells from a non-embryonic source.
An advantage of this invention is the extraction of up to twice as many stem cells from a placenta or umbilical cord than that achieved by simply draining these organs.